Nitric oxide synthase distribution and expression with ischemic preconditioning of the rat liver (original) (raw)
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Role of endothelial nitric oxide synthase in remote ischemic preconditioning of the mouse liver
Liver Transplantation Official Publication of the American Association For the Study of Liver Diseases and the International Liver Transplantation Society, 2011
Hindlimb remote ischemic preconditioning (RIPC) reduces liver ischemia/reperfusion (IR) injury in wild-type mice. The underlying mechanisms of RIPC are currently unknown. In this study, we investigated the role of endothelial nitric oxide synthase (eNOS) in mediating the protective effects of RIPC. Endothelial nitric oxide synthase knockout (eNOS À/À) mice were divided into 4 groups: (1) a sham surgery group, (2) an RIPC group (6 cycles of 4 minutes of hindlimb ischemia and 4 minutes of hindlimb reperfusion), (3) an IR group [40 minutes of lobar (70%) hepatic ischemia and 2 hours of reperfusion], and (4) an RIPCþIR group (RIPC followed by the IR group procedures). Plasma liver aminotransferases, hepatic histopathological injury scores, transmission electron microscopy studies, and hepatic microcirculatory blood flow (MBF) were assessed. eNOS protein expression was analyzed in the livers and hindlimb muscles of wild-type mice. Hindlimb RIPC did not protect against subsequent liver IR injury in eNOS À/À mice; this was demonstrated by the lack of reduction in the plasma aminotransferase levels, histopathological scores, or ultrastructural features of IR injury in the RIPCþIR group versus the IR group. Hepatic MBF did not recover during liver reperfusion in the RIPCþIR group versus the IR group. eNOS protein expression was similar among all wild-type groups. In conclusion, eNOS is essential for the protective effects of hindlimb RIPC on liver IR injury. eNOS exerts its protective effects through the preservation of hepatic MBF. At 2 hours of reperfusion, eNOS protection is likely due to the increased activation of eNOS rather than increased expression. Liver Transpl 17:610-619, 2011. V C 2011 AASLD.
Clinical Science, 2011
NO (nitric oxide) may protect the liver from IR (ischaemia/reperfusion) injury. RIPC (remote ischaemic preconditioning) also protects against liver IR injury; however, the molecular mediator(s) of RIPC are currently unknown. The aim of the present study was to assess the role of NO in hindlimb RIPC-induced protection against liver IR injury. Mice were allocated to the following groups: sham group; RIPC group (six cycles of 4×4 min IR of hindlimb); IR group [40 min lobar (70%) hepatic ischaemia and 2-h reperfusion]; RIPC+IR group (RIPC followed by IR group procedures); and C-PTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt]+RIPC+IR group [C-PTIO (a direct NO scavenger) was administered, followed by the RIPC+IR group procedure]. Hepatic MBF (microcirculatory blood flow) was measured throughout the experiment. Circulating NOx (nitrite and nitrate) levels, plasma liver transaminases, hepatic histopathological and TEM (transmission electron microscop...
L-arginine in the ischemic phase protects against liver ischemia-reperfusion injury
Acta Cirurgica Brasileira, 2012
PURPOSE: To investigate the effects of intravenous L-arginine (LG) infusion on liver morphology, function and proinflammatory response of cytokines during the early phase of ischemia-reperfusion injury (IRI). METHODS: Thirty rabbits were subjected to 60 minutes of hepatic ischemia and 120 minutes of reperfusion. An intravenous injection of saline or L-arginine was administered five minutes before the ischemia and five minutes before initiating the reperfusion and at the 55 th and 115 th minutes after the ischemia. Samples were collected for histological analysis of the liver and measurements of the serum AST, ALT and LDH and the cytokines IL-6 and TNF-alpha. RESULTS: It was observed a significant reduction of sinusoidal congestion, cytoplasmic vacuolization, infiltration of polymorphonuclear leukocyte, nuclear pyknosis, necrosis and steatosis in liver tissue, as well as AST, ALT and LDH after injection of LG in the ischemia (p <0.001). Lower levels of IL-6 and TNF-alpha were associated with LG infusion during ischemia. Higher levels these proteins were observed in animals receiving LG during reperfusion. CONCLUSION: L-arginine protects the liver against ischemia/reperfusion injury, mainly when is administered during the ischemic phase.
Liver International, 2012
Ischaemia reperfusion (IR) injury is a clinical entity with a major contribution to the morbidity and mortality of liver surgery and transplantation. A central pathway of protection against IR injury utilizes nitric oxide (NO). Nitric oxide synthase (NOS) enzymes manufacture NO from L-arginine. NO generated by the endothelial NOS (eNOS) isoform protects against liver IR injury, whereas inducible NOS (iNOS)-derived NO may have either a protective or a deleterious effect during the early phase of IR injury, depending on the length of ischaemia, length of reperfusion and experimental model. In late phase hepatic IR injury, iNOS-derived NO plays a protective role. In addition to NOS consumption of L-arginine during NO synthesis, this amino acid may also be metabolized by arginase, an enzyme whose release is increased during prolonged ischaemia, and therefore diverts L-arginine away from NOS metabolism leading to a drop in the rate of NO synthesis. NO most commonly acts through the soluble guanylyl cyclase-cyclic GMPprotein kinase G pathway to ameliorate hepatic IR injury. Both endogenously generated and exogenously administered NO donors protect against liver IR injury. The beneficial effects of NO on liver IR are not, however, universal, and certain conditions, such as steatosis, may influence the protective effects of NO. In this review, the evidence for, and mechanisms of these protective actions of NO are discussed, and areas in need of further research are highlighted.
Journal of Surgical Research, 2014
Background: ischemic preconditioning (IPC) protects against liver ischemiaereperfusion (IR) injury. The mechanism involves nitric oxide metabolism but the importance of endothelial nitric oxide synthase (eNOS) has not been established. Heme oxygenase-1 (HO-1) protects against liver IR but it is unclear if this depends on nitric oxide synthase. Materials and methods: A mouse model of IPC with liver IR using wild-type (WT) and eNOS transgenic knockout (eNOSÀ/À) mice was developed to study the role of eNOS and its relationship to HO-1. Serum alanine aminotransferase level, liver histopathologic injury scores, and liver microcirculatory blood flow were measured. Western blots measured liver HO-1/2, eNOS, phosphorylated eNOS, inducible nitric oxide synthase, and reverse transcriptionepolymerase chain reaction (HO-1). A set of 24-h recovery experiments was undertaken on WT mice with measurement of serum alanine aminotransferase level, histologic injury score, and HO-1 by Western blot. Results: In WT animals, IPC preceding IR resulted in a reduction in hepatocellular and histologic injury, and improvement in parenchymal perfusion. In contrast, IPC in the eNOSÀ/À model did not protect the animals from IR injury. There was no difference between the eNOS and phosphorylated eNOS expression in all the WT groups. HO-1 protein was not detected in the nonrecovery groups but HO-1 messenger RNA was detected in all groups. In WT recovery experiments, IPC was protective against IR injury. HO-1 protein was detected in the IPC þ IR and IR only groups but not in the sham group. Conclusions: This study developed and used an eNOSÀ/À model to demonstrate that eNOS mediates protection against liver IR injury by IPC. The eNOS expression and activity and HO-1 expression are increased independently in liver IPC and IR, with HO-1 expression increased in the later stages of IPC and IR.
Background: Both pre-and postconditioning have been shown to protect the liver parenchyma from ischemia/ reperfusion (I/R) injury during hepatectomy by altering the production of NO. However, to date there is no study to compare their effect on the inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) gene expression, who are the main modulators in the pathway of NO during the acute phase of I/R injury. Methods: We designed a prospective experimental cohort comprising of three groups (sham group-SG, preconditioning-PrG and postconditioning group-PoG) and consisting of 10 animals per group. All animals underwent extended hepatectomy (70%) under prolonged warm ischemia either after preconditioning or followed by postconditioning or without any protective maneuver (SG). Following reperfusion blood samples and liver biopsies were obtained at the start of reperfusion (0 hours), at 6 and 12 hours post reperfusion. iNOS and eNOS gene expression was assessed on liver tissue by polymerase chain reaction (PCR); in addition, the extent of hepatocellular injury was histologically assessed. Results: At the beginning of reperfusion iNOS expression was significantly reduced in the PoG in comparison to the SG (Kruskal-Wallis test, P=0.012; Mann-Whitney U test, P<0.0005 Bonferroni correction) and continued to remain at low levels until 6 hours post reperfusion (Kruskal-Wallis test, P=0.01; Mann-Whitney U test, P<0.0005-Bonferroni correction) This difference was eliminated by 12 hours. No significant differences were found in the expression of eNOS between groups and within time measurements. Aspartate aminotransferase (AST) and Alkaline phosphatase (ALP) were found increased at the start of reperfusion; their levels continued to increase by 6 hours in all groups, however only in the PoG the increase attended statistical significance at 12 hours after reperfusion. ALT levels presented only minor alterations during the course of reperfusion. The PrG was found to have more intense hepatocellular injury at the start of reperfusion than the PoG however, that appeared to gradually settle by 12 hours in contrast to PoG where the hepatocellular injury continued to deteriorate. Conclusions: PoG appeared to decrease iNOS overexpression more effectively than PrG in comparison to animals who have undergone no protective maneuver (SG). However, PrG was more effective than PoG in ameliorating the hepatocellular injury observed at 12 hours after the ischemic insult.
International Journal of Medicine and Medical Research, 2014
Background. Hepatic ischemia-reperfusion (I/R) injury occurs upon restoration of hepatic blood flow after a period of ischemia. Objective. The study establishes that stimulation or blockade of nitric oxide synthesis has a protective effect during ischemia-reperfusion. Methods. Male albino rats which were divided into four equal groups: sham-operated control, ischemia and reperfusion group (0.9 % saline i.p.) for 3 days, group pre-treated with L-arginine (25 mg/kg i.p.), group pre-treated with L-NAME (10 mg/kg i.p.) for 3 days before ischemia-reperfusion maneuver. Complete ischemia of the median and left hepatic lobes was induced by clamping the left branches of the portal vein and the hepatic artery for 45 min. Rats were sacrificed after 3-h reperfusion. Nitric oxide synthase 3 (endothelial) and nitric oxide synthase 2 (inducible) expression, nitric oxide stabile metabolites (NO 2 , NO 3) content, AST and ALT activities were determined. Histological examination of liver tissue was performed. Conclusions. Relative NO deficiency, due to eNOS inhibition, is central in the pathogenesis of hepatic ischemia reperfusion injury. Replacing NO content with either precursors or via donor drugs represents novel methods in ameliorating ischemia-reperfusion injury.
Liver Ischemic Preconditioning Is Mediated by the Inhibitory Action of Nitric Oxide on Endothelin
Biochemical and Biophysical Research Communications, 1996
The concerted involvement of both NO and endothelin in the protective effect of preconditioning against hepatic ischemia-reperfusion induced injury has been evaluated in this study. Thus hepatic ischemiareperfusion or preconditioning plus ischemia-reperfusion was induced in rats and the effect of nitric oxide administration or inhibition with addition of the endothelin antagonist Bosentan was evaluated. Results show that the increases in plasma GPT release after ischemia-reperfusion were prevented after preconditioning. Inhibition of nitric oxide abolished the effect of preconditioning, addition of the endothelin antagonist abolished the injurious effect of NO inhibition. Also, increased synthesis of endothelin has been detected after ischemia-reperfusion, and addition of NO or preconditioning prevented this increase, suggesting that increases of NO inhibit endothelin synthesis. Altogether this indicates that hepatic preconditioning is mediated by the inhibitory action of nitric oxide on endothelin levels.